Thrombus formation is essential for preventing blood loss and allowing repair of an injured vessel, a process known as hemostasis, yet a thrombus can also be pathologic when it occludes a blood vessel depriving tissue of oxygen. The occlusion of an artery by a thrombus, arterial thrombosis, most often occurs at the site of a ruptured or eroded atherosclerotic plaque (Kou, V. et al., (2006) Mt. Sinai J. Med. 73: 449-468). Specific occlusion of the coronary arteries results in acute coronary syndrome which includes unstable angina and myocardial infarction (MI).
A fibrin clot may be produced in blood by initiation of one of two distinct routes, the intrinsic and extrinsic pathways, which converge onto a common pathway of coagulation (Macfarlane, R. G. (1964) Nature 202: 498-9; Davie, E. W. et al. (1964) Science 145: 1310-12; Joseph, K. et al. (2005) Advances Immunology 86: 159-208). Experimental data have suggested both PK- and FXII-deficient individuals have severely impaired intrinsic pathway-mediated clot formation despite their lack of bleeding phenotype (Ratnoff, O. D. et al. (1955) J. Clin. Invest. 34: 602-613; Colman, R. W. (2001) In Hemostasis and Thrombosis: Basic principles and clinical practice. R. W. Colman et al eds. Lippincott Williams & Wilkins, Philadelphia, Pa. 103-122; Rosen, E. D. et al. (1997) Nature 390: 290-294; Hathaway, W. E., et al. (1965) Blood 26: 521-32; Lawrie, A. S. et al (1998) Clin. Lab. Haematol. 20: 179-86; and Bates, et al., (2005) Circulation 112: 53-60). In the intrinsic pathway, by binding to the surface, a small amount of factor XII (FXII) is activated (FXIIa) which in turn activates plasma kallikrein (PK) through proteolysis. Importantly, PK then generates additional FXIIa in a feedback loop which in turn activates factor XI (FXI) to FXIa to connect to the common pathway. Although the initial activation of the intrinsic pathway is through a small amount of FXIIa activating a small amount of PK, it is the subsequent feedback activation of FXII by PK that controls the extent of activation of the intrinsic pathway and hence downstream coagulation (Hathaway, W. E., et al. (1965) Blood 26: 521-32).
Current treatment for acute MI or ischemic stroke in a hospital setting requires emergency measures to dissolve the occluding thrombus and allow reperfusion (restored blood flow). One of the common ways of doing this is by treating the patients with fibrinolytic agents, such as tissue plasminogen activator (t-PA) or streptokinase, agents that lead to the generation of active plasmin from plasminogen. Plasmin cleaves the fibrin meshwork of the thrombus, therefore leading to clot dissolution. Such fibrinolytic agents are the most frequently used treatment for reperfusion worldwide. However, fibrinolysis is also associated with a high degree of re-thrombosis with subsequent rates of reocclusion of up to 50% depending on the study (Zijlstra, F. et al (1993) N. Engl. J. Med. 328: 680-4; Brodie, B. R. et al. (1994) Circulation 90: 156-62; Stone, G. W. et al (1999) Circulation 99: 1548-54; Tamai, H. et al, MAJIC Investigators (2004) Am. Heart J. 147: E9; Verheugt, F. W. et al (1996) J. Am. Coll. Cardiol. 27: 766-73).
Patients who have undergone acute MI show clinical evidence of being in a hypercoagulable (clot-promoting) state. This hypercoagulability is paradoxically additionally aggravated in those receiving fibrinolytic therapy. Increased generation of thrombin, as measured by thrombin-antithrombin III (TAT) levels up to 2-fold higher, is observed in patients undergoing such treatment compared to the already high levels observed in those receiving heparin alone (Hoffmeister, H. M. et al (1998) Circulation 98: 2527-33). The increase in thrombin has been proposed to result from plasmin-mediated activation of the intrinsic pathway. Plasmin-mediated activation of the intrinsic pathway system is known to occur in blood (Ewald, G. A. et al. (1995) Circulation 91: 28-36), and it has been suggested that this occurs as a consequence of direct activation of FXII by plasmin.
Not only does the fibrinolysis-induced hypercoagulability lead to increased rates of reocclusion, it is also probably responsible, at least in part, for failure to achieve complete fibrinolysis of the clot, a major shortcoming of fibrinolytic therapy (Keeley, E. C. et al. (2003) Lancet 361: 13-20). Another problem in fibrinolytic therapy is the accompanying 3-fold elevated risk of intracranial hemorrhage (ICH) (Menon, V. et al (2004) 126: 549S-575S; Fibrinolytic Therapy Trialists' Collaborative Group (1994) Lancet 343: 311-322). Hence, an adjunctive anti-coagulant therapy that does not increase the risk of bleeding, but inhibits the formation of new thrombin, would be greatly beneficial.
It has been found that treatment of wild-type mice with an irreversible inhibitor of FXII led to fewer occluded vessels and less ischemic cortical damage and inhibition of FXII, would be protective for arterial thrombosis, such as that occurring during acute MI or during thrombotic stroke (WO/2006 066878 A1). However, peptidic drugs have numerous shortcomings including limited application to acute studies because of short half lives, i.v. administration requiring medical intervention, and the development of anti-peptide antibodies by patients undergoing treatment.
Therefore, there is a need to develop small molecule inhibitors of PK that will overcome these limitations. In particular, inhibitors that can push the balance of fibrinolysis/thrombosis at the occluding thrombus toward dissolution, promote reperfusion and also attenuate the hypercoagulability state, thus preventing the thrombus from reforming and reoccluding the vessel. The present invention fulfills this and other needs.